Expression of ciliary neurotrophic factor (CNTF) and its tripartite receptor complex by cells of the human optic nerve head.

PURPOSE
Ciliary neurotrophic factor (CNTF) promotes gene expression, cell survival and differentiation in various types of peripheral and central neurons, glia and nonneural cells. The level of CNTF rises rapidly upon injury to neural tissue, suggesting that CNTF exerts its cytoprotective effects after release from cells via mechanisms induced by cell injury. The purpose of this study was to determine if cells in the optic nerve head express CNTF and its tripartite receptor complex.


METHODS
Well-established optic nerve head astrocytes (ONHA) and lamina cribrosa (LC) cell cultures were derived from normal human donors. Total RNA was reverse transcribed and polymerase chain reaction (PCR) amplified for mRNA detection. Cytoplasmic protein expression was determined by immunocytochemistry and Western blot analysis of the cellular lysates. Serum free medium was concentrated and used for detecting extracellular proteins. CNTF complexed with CNTFR-alpha was assayed by immunoprecipitation.


RESULTS
Cells isolated from the human optic nerve head express CNTF and its tripartite receptor complex members (CNTFR-alpha, gp130, LIFR-beta).


CONCLUSIONS
Taken together, these data suggest a possible neuroprotective role of CNTF in the optic nerve head.

Total cellular RNA extraction and cDNA synthesis: Total cellular RNA from 1x10 7 cells was prepared using the TRIzol reagent (Invitrogen Life Technologies, Carlsbad, CA). After isopropanol precipitation, the RNA was resuspended in 20 µl of water and stored at -80 °C. First strand complementary DNA (cDNA) synthesis from total cellular and tissue RNA and details of PCR procedure were prepared as previously described [23].
Immunocytochemical localization of ciliary neurotrophic factor and its receptor complex: Immunocytochemistry studies of CNTF and its receptor complex in LC cell lines and ONHA were performed as previously described [23]. Fluorescence was detected using a Nikon Microphot-FXA microscope with the appropriate filter. Images were recorded and processed using IPLabs 5.0 software (Scanalysis, Inc., Fairfax, VA). Control immunohistochemical preparations included both (a) omission of the primary antibody and (b) neutralization of the primary antibody with a 10-fold (by weight) excess of blocking peptide (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) in PBS overnight at 4 °C.
Western blot analysis of ciliary neurotrophic factor and Its receptor complex: Proteins from confluent or near confluent LC cells, and ONHA (1x10 7 ) were extracted and assayed via Western blot analysis as previously reported [23]. Omission of the primary antibodies served as negative controls. Western blots were repeated twice to confirm results. After the final wash, membranes were treated with enhanced chemiluminescence (ECL) Western blotting kit detection reagents (Amersham Pharmacia Biotech, Piscataway, NJ) and exposed to Hyperfilm ECL (Amersham Pharmacia Biotech) for the period of time ranging from 1 s to 30 min depending on the amount of target protein. Some of the images were also captured using the computerized charge-coupled device (CCD) camera-based imaging system (Alpha Innotech, San Leadro, CA) following application of SuperSignal West Femto Maximum Sensitivity Substrate (Pierce Biotechnology, Inc. Rockford, IL) on the membranes.
Immunoprecipitation: Human ONHA and LC cells (1x10 7 cells) were grown in serum-free culture medium for 48 h. The serum-free medium was collected and concentrated 20-fold using centricon YM-3 (Millipore Corporate, Billerica, MA).   Cellular protein was collected in lysis buffer (150 mM NaCl, 1% Triton X-100, 50 mM Tris). A protease inhibitor cocktail (Sigma-Aldrich, St. Louis, MO) was added to each sample upon collection. Protein concentration was measured using the Bio-Rad Dc Protein Assay System (Bio-Rad Laboratories, Richmond, CA). Cellular lysate (100 µg) was incubated with 5 µg goat anti-CNTFR-α IgG overnight. Samples were then incubated with 50 µl of 50% protein G-agarose for 2 h at 4 °C, washed 4 times, and resuspended in sample buffer. Immunoprecipitated proteins were separated on 10% denaturing polyacrylamide gels and electrophoretically transferred to nitrocellulose membranes. Blots were blocked and analyzed as described in Western blot analysis section above.

RESULTS
Expression of ciliary neurotrophic factor and ciliary neurotrophic factor tripartite receptor complex mRNAs in human lamina cribrosa cells and optic nerve head astrocytes: The mRNA expression of CNTF, CNTFR-α, gp130, and LIFR-β from LC cells and ONHA are represented as PCR products in Figure 1. In all cell lines tested, including a brain astrocyte cell line that is known to express CNTF and its receptor complex, a single band at the correct size was detected following gel electrophoresis. This PCR product is unlikely a genomic DNA contamination, as a set of β-actin primers that span an intron did not amplify a genomic fragment. Additionally, in an optic nerve head tissue sample (Lane 9), the presence of a PCR product confirmed the in vivo expression of CNTF and the receptor complex. Therefore, human LC cells and ONHA express mRNA for CNTF and its tripartite receptor complex (i.e. CNTFR-α, gp130 and LIFR-β). Figure 2 demonstrates the protein expression of CNTFR-α, gp130, and LIFR-β in human LC cells and ONHA by the Western blot analysis. For each protein, the specific antibody recognized a single band from both cell types. Without the primary antibody, no band was recognized on a gel that was run and analyzed in parallel, which ruled out the nonspecific reactivity of the secondary antibody. In agreement with the literature [24,25], we detected a 52.3 kDa protein for CNTFRα and a 190-210 kDa protein for LIFR-β in both cell types. Gp130 stained as a canonical 130-150 kDa protein in LC cells [24,26]. Despite our numerous attempts, we were unable to detect CNTF protein in either LC cells or ONHA cell lysate (data not shown).

Expression of ciliary neurotrophic factor and the ciliary neurotrophic factor tripartite receptor complex proteins in human lamina cribrosa cells and optic nerve head astrocytes:
Complementary to the Western blot analysis, our immunocytochemistry study localized CNTFR-α, gp130, and LIFRβ proteins in both LC cells and ONHA from multiple donors in vitro. A representative picture of each receptor component in each cell type is shown in Figure 3. All members of the CNTF receptor complex were localized in both cell types. The staining pattern for all three components appeared punctate, resembling typical membrane proteins. There were no obvious differences in either intensity or staining pattern between LC cells and ONHA. When primary antibodies were omitted or blocking peptide neutralization was implemented, no staining was observed. There was no detectable fluorescent signal for CNTF in either cell type (data not shown). This is in agreement with previous literature that CNTF is detectable only at very low levels in the intact adult central nervous system (CNS).
Ciliary neurotrophic factor-ciliary neurotrophic factor receptor-α complex protein expression by lamina cribrosa cells and optic nerve head astrocytes: A CNTF-CNTFR-α protein complex was detected in LC cells and ONHA, although expression of CNTF was at extremely low levels. The cell lysates and the serum-free culture medium of both cell lines were concentrated and subjected to immunoprecipitation with a specific antibody against CNTFR-α. The resultant mixture was then enriched with CNTFR-α and its complexes. CNTF-CNTFR-α complexes in all samples were revealed by western immunoblotting using a specific antibody against CNTF (Figure 4). From both cell lysates and conditioned media, we were able to detect a single CNTF protein band of appropriate molecular weight. Our results detected the expression of CNTF protein in complex with CNTFR-α in both LC cells and ONHA. The necessity of extensive concentration procedures supported the previous notion that CNTF protein expression is normally extremely low.

DISCUSSION
Our study provides direct evidence that cultured LC cells and ONHA constitutively express CNTF and its tripartite receptor complex. The presence of the CNTF receptor complex enables both LC cells and ONHA to respond to CNTF. The ONH harbors LC cells and ONHA, which can be potential sources of CNTF during stressful events [1,10,21,27]. Therefore, CNTF can signal ONH cells in a paracrine as well as autocrine fashion. Consistent with previous reports in the brain, CNTF is expressed at low levels in these ONH cell types under normal conditions. As a member of the IL-6 growth factor family, CNTF shares some of the receptor components with the family members in forming a multi-component signal-transducing receptor complex. Presumably, there are overlapping biological activities and a degree of functional redundancy between CNTF and other family members. Conceivably, IL-6 family members other than CNTF may maintain the physiological function of the ONH through the receptors under normal conditions. Our study raises the possibility of a neuroprotective role of CNTF in human optic nerve head via a paracrine/autocrine mechanism.
Since CNTF does not contain a classical signal sequence for secretion, it has long been postulated that CNTF are released by cytolysis due to injury. A recent study of bovine corneal epithelial cells [28] suggested that CNTF may be externalized while binding to CNTFR-α. As a glycosylphosphatidylinositol (GPI)-anchored coreceptor, CNTFR-α can carry CNTF to the outer leaflets of the cell membrane and be released to the extracellular space upon phospholipase cleavage. This theory is not completely novel, as cardiotropin-like cytokine (CLC), a member of the CNTF neurotrophic factor family, generates a similar functional composite cytokine with CNTFR-α (CLC-CNTFR-α complex) in mammalian cells [29]. Our results support this type of mechanism. First of all, the immunoprecipitation study demonstrated that CNTF-CNTFR-α complex is present extracellularly, indicating that CNTF and its α receptor are released as a complex. Secondly, under normal conditions, there is minimal cytolysis, and the CNTF released by disintegration of cells is below the threshold of detection. Therefore, the presence of CNTF in the cell culture medium is more than likely due to externalization and release of the CNTF-CNTFR complex instead of cytolysis.
In conclusion, our study demonstrates the potential for a significant signaling system using CNTF family neurotrophic factors in glial cells of the optic nerve head. In addition to the current study, we have also reported that neurotrophin signaling systems in LC cells, ONHA, and ONH tissue [1]. We have also reported the presence of glial-derived neurotrophic factor (GDNF) and the receptor complex in the same cell types [27]. Neurotrophic factors and neurotrophins may play a critical role in restoring the normal function of lamina cribrosa, especially during the time of injury. This implies that neurotrophin and neurotrophic factors may have therapeutic applications for neuroprotection in glaucoma patients.